The present invention relates to non-slow-binding thrombin inhibitors, a process for the preparation of said inhibitors, pharmaceutical compositions containing the same, and the use of these thrombin inhibitors as antithrombotic agents.
Much attention has been focused on inhibition of thrombin as potential anticoagulants. Inhibitors of the enzyme thrombin, a key serine protease within the blood coagulation cascade, have for some time been considered as potential candidates for anticoagulant prophylaxis and therapy. In particular the multiple roles played by thrombin in its actions on coagulation factors, circulating blood components, and the cells of the vessel wall makes it a particularly attractive target in a variety of pathological states. Moreover, limitations associated with currently employed anticoagulants, in particular the occurrence of bleeding complications, necessitate the search for more specifically-acting agents.
Many peptide(-like) serine protease inhibitors have been disclosed, amongst which are transition state inhibitors of thrombin. Many of these latter compounds, however, are slow-binding inhibitors. The use of slow-binding inhibitors of thrombin is open to criticism. In vivo, thrombin is constantly generated in plasma and thrombin inhibitors primarily function by slowing thrombin formation through inhibiting thrombin-mediated amplification steps. To slow down such an amplification cascade, a non-slow-binding inhibitor would be preferable. A larger dose of a slow-binding inhibitor would be needed to achieve the same effect, with a correspondingly increased risk of side-effects.
Relevant thrombin inhibitors are disclosed by Brady et al., Bioorganic and Medicinal Chemistry, 3 (1995), 1063-78 wherein D-Phe-Pro-Arg-amide and D-Phe-Pro-Lys-X derivatives have been disclosed, X being a ketoester or amine. These compounds are disclosed to be slow-binding thrombin inhibitors, and likewise these compounds are excluded from the present invention. In the search for non-slow-binding thrombin inhibitors Jones et al., J. Enzyme Inhibition, 9 (1995), 43-60 attempted to obtain improvement by using D-Cha-Pro-Lys-COOH derivatives. However, although these derivatives proved to be more potent thrombin inhibitors, they still exhibit slow-binding properties.
In a recent attempt to obtain potent non-slow-binding thrombin inhibitors Lewis et al., Thrombosis and Haemostasis, 74(4) (1995), 1107, prepared Me-D-Phe-Pro-Lys-X derivatives, X being carboxyamide or carboxylic acid. These compounds, among which specifically disclosed Me-D-Phe-Pro-Lys-COOH, are classified as slow-binding inhibitors. This compound therefore does not fulfill the requirements of the present invention and is excluded from protection.
A thrombin inhibitor with an alkyl-substituted lysine is disclosed in U.S. Pat. No. 5,523,308. In earlier references other Phe-Pro-Lys sequences are described, for example by Iwanowicz et al. in Bioorganic and Medicinal Chemistry Letters, 2 (1992), 1607-12, which discloses D-Phe-Pro-Lys-X derivatives, X being i.a. a ketoester. Such compounds may also be described as slow-binding thrombin inhibitors.
Other types of peptides for inhibition of different serine proteases are also disclosed. Tsutsumi et al. in J. Med. Chem., 37 (1994), 3492-3502 described peptide-like compounds having thiazole and benzothiazole C-terminal ends. It was found that such thiazole derivatives are 300 times more potent than the corresponding thiophene analogues. It was further posited that C-terminal heterocyclic groups would provide a critical hydrogen-bond interaction with the histamine of the protease prolyl endopeptidase. Although it was further suggested that this feature may well be capable of extension to other serine proteases, thrombin proteases were not specifically mentioned. The mechanistic explanation of Tsutsumi was later challenged by Edwards et al. in J. Med. Chem., 38 (1995), 76-85, but also these authors found that elastase inhibitors of the type D-Phe-Val-Pro-Val-X, X being thiazole and benzothiazole, are non-slow-binding inhibitors of the relevant serine protease. These authors suggest the development of peptidyl xcex1-ketoheterocycles as inhibitors of other serine proteases as well.
The present invention relates to the surprising finding that the teachings of Edwards, Tsutsumi and others can also be applied to thrombin inhibitors. The application of the C-terminal heterocycles to the compounds as disclosed by Lewis, Jones and Brady provide potent thrombin inhibitors having non-slow-binding properties to thrombin. Moreover, many of these compounds show improved biological half-lifes and oral bioavailability.
The invention therefore relates to non-slow-binding thrombin inhibitors of the formula:
Axe2x80x94Bxe2x80x94C-Lys-D
wherein
A is H, 2-hydroxy-3-cyclohexyl-propionyl-, R1, R1xe2x80x94Oxe2x80x94CCxe2x80x94, R1xe2x80x94COxe2x80x94 , R1xe2x80x94SO2xe2x80x94, xe2x80x94(CHR2)nCOOR3, or an N-protecting group, wherein
R1 is selected from xe2x80x94(1-6C)alkylene-COOH, (1-12C)alkyl, (2-12C)alkenyl, (6-14C)aryl, (7-15C)aralkyl and (8-16C)aralkenyl, the aryl group of which may be substituted with (1-6C)alkyl, (2-12C)alkoxy, hydroxy, or halogen;
R2 is H or has the same meaning as R1;
R3 is selected from H, (1-12C)alkyl, (2-12C)alkenyl, (6-14C)aryl, (7-15C)aralkyl and (8-16C)aralkenyl, the aryl group of which may be substituted with (16C)alkyl, (2-12C)alkoxy, hydroxy, or halogen;
n is an integer of 1 to 3;
B is a bond, L-Asp or an ester derivative thereof, Leu, norLeu, xe2x80x94N(benzyl)xe2x80x94CH2xe2x80x94COxe2x80x94, xe2x80x94N(2-indane)xe2x80x94CH2xe2x80x94COxe2x80x94, D-1-Piq, D-3-Piq, D-Tiq, Atc or a D-amino acid having a hydrophobic aromatic side chain;
C is Azt, Pro, Pec, norLeu(cyclo)Gly, or an amino acid of one of the formulae xe2x80x94N[(3-8C)cycloallkyl]-CH2xe2x80x94COxe2x80x94 or xe2x80x94N(benzyl)-CH2xe2x80x94COxe2x80x94;
D is selected from COOH, tetrazole, oxazole, thiazole and benzothiazole;
or A and C have the aforesaid meanings, B is Dxe2x80x94(3-8C)cycloalkylalanine, and D is tetrazole, oxazole, thiazole or benzothiazole;
or a prodrug thereof,
or a pharmaceutically acceptable salt thereof;
with the exception of the compound Me-D-Phe-Pro-Lys-COOH.
The compounds of the present invention are useful for treating and preventing thrombin-mediated and thrombin-associated diseases. This includes a number of thrombotic and prothrombotic states in which the coagulation cascade is activated which include, but are not limited to, deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterial occlusion from thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, restenosis following arterial injury or invasive cardiological procedures, postoperative venous thrombosis or embolism, acute or chronic atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention may also be used as anticoagulants in extracorporeal blood circuits, as necessary in dialysis and surgery. The compounds of the invention may also be used as in vitro anticoagulants.
Preferred compounds according to this invention are the compounds wherein D is COOH. In addition, preferably A is H, (1-12C)alkyl, xe2x80x94COxe2x80x94(7-15C)aralkyl, xe2x80x94SO2xe2x80x94(1-12C)alkyl, xe2x80x94SO2xe2x80x94(6-14C)aryl, or xe2x80x94SO2(7-15C)aralkyl; B is a bond, L-Asp, norLeu, D-1-Piq, or D-Phe; and C is Pro, norLeu(cyclo)Gly, or xe2x80x94N-cyclopentylxe2x80x94CH2xe2x80x94COxe2x80x94. More preferred are the non-slow-binding thrombin inhibitors wherein A is xe2x80x94SO2-benzyl, B is a bond, and C is norLeu(cyclo)Gly, or wherein A is xe2x80x94SO2-ethyl, B is D-Phe, and C is Pro; or wherein A is hydrogen, B is D-1-Piq, and C is Pro.
Other preferred compounds according to the invention are those wherein D is oxazole or thiazole. Further, preferably A is H, (1-12C)alkyl, 2-hydroxy-3-cyclohexyl-propionyl-, xe2x80x94COxe2x80x94(CH2)nCOOH, xe2x80x94COxe2x80x94(7-15C)aralkyl, xe2x80x94SO2xe2x80x94(6-14C)aryl, xe2x80x94SO2xe2x80x94(7-15C)aralkyl, xe2x80x94SO2xe2x80x94(1-12C)alkyl, xe2x80x94(CHR2)nCOOR3, R2 being H or (1-12Calkyl) and R3 being H, (1-12C)alkyl or benzyl; and C is Pro, norLeu(cyclo)Gly, or xe2x80x94N[(3-8C)cycloalkyl]-CH2xe2x80x94COxe2x80x94. Particularly preferred are the non-slow-binding thrombin inhibitor wherein A is xe2x80x94(CH2)nCOOR3, R3 being H, (1-12C)alkyl or benzyl; B is Dxe2x80x94(3-8C)cycloalkylalanine, or D-Phe optionally monosubstituted with alkoxy or halogen; and C is Pro. The most preferred compounds of the invention are compounds wherein D is thiazole. Specifically preferred is the non-slow-binding inhibitor HOOCxe2x80x94CH2D-Cha-Pro-Lys-(2-thiazolyl).
The N-protecting group as defined in the definition of moiety A is any N-protecting group as used in peptides. Suitable N-protecting groups can be found in T. W. Green and P. G. M. Wuts: Protective Groups in Organic Synthesis, Second Edition (Wiley, N.Y., 1991) and in The Peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, Eds., (Academic Press, New York, 1981).
Alkyl, as used herein, is a branched or unbranched alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, isopentyl, dodecyl, and the like.
The term (1-6C)alkylene means a branched or unbranched alkylene group having 1 to 6 carbon atoms, such as xe2x80x94(CH2)mxe2x80x94 and m is 1 to 6, xe2x80x94CH(CH3)xe2x80x94, xe2x80x94CH(CH3)xe2x80x94(CH2)xe2x80x94, etc. The preferred alkylene group is methylene.
Alkenyl is a branched or unbranched unsaturated alkenyl group having 2 to 12 carbon atoms. Examples are ethenyl, propenyl, allyl, and the like.
Aralkyl and aralkenyl groups are alkyl and alkenyl groups respectively, substituted by one or more aryl groups, the total number of carbon atoms being 7 to 15 and 8 to 16, respectively. Preferred aralkyl groups are e.g. of the formulae xe2x80x94(CH2)pxe2x80x94CHxe2x80x94(C6H5)2, p being 1 or 2, or xe2x80x94CH2)qxe2x80x94C6H5 optionally substituted with halogen, q being 1,2 or 3.
Aryl in above-mentioned definition and in the definition of aryl, as used in the compound of the invention, is an aromatic moiety of 6 to 14 carbon atoms. The aryl group may further contain one or more hetero atoms, such as N, S, or O. Examples of aryl groups are phenyl, naphthyl, (iso)quinolyl, indanyl, and the like. Most preferred is the phenyl group. The aryl group may be substituted with on or more alkyl groups, preferably methyl, alkoxy groups, preferably methoxy, hydroxy, or halogen. The term halogen means fluorine, chlorine, bromine or iodine. Chlorine is the preferred halogen.
The terms D-1-Piq and D-3-Piq mean 1- and 3-carboxyperhydroisoquinoline, respectively. The term Tiq means 1,2,3,4-tetrahydroisoquinoline-carboxylic acid. Atc is 2-aminotetralin-2-carboxylic acid. The terms Azt and Pec mean 2-azetidine carboxylic acid and pipecolinic acid, repectively.
The term norLeu(cyclo)Gly means a structural fragment of the formula 
The term hydrophobic aromatic side chain means a (1-12C)alkyl substituted with one or more (6-14C)aryl groups (which may contain a heteroatom, e.g. nitrogen) such as phenyl, pyridinyl, naphthyl, tetrahydronaphthyl, and the like, which hydrophobic side chain may be substituted with hydrophobic substituents such as halogen (preferably chlorine), trifluoromethyl, lower alkyl (for instance methyl or ethyl), lower alkoxy (for instance methoxy), phenyloxy, benzyloxy, and the like.
The term (3-8C)cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.
Tetrazole, oxazole, thiazole and benzothiazole have the following formulae, respectively: 
The invention also includes prodrugs of the compounds of the general formula, which after administration are metabolized into the active compounds. Suitable prodrugs are for example N-alkoxycarbonyl protected (preferably N-ethoxycarbonyl) derivatives of the general formula.
As used herein the term pharmaceutically acceptable salt refers to salts that retain the desired biological activity of the parent compound and preferably do not impart any undesired toxic effects. Examples of such salts are acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid nitric acid, and the like. Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. Salts may be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like, or with an organic cation formed from N,Nxe2x80x2-dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g. a zinc tannate salt).
The compounds of this invention possess one or more chiral carbon atoms, and may therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g. crystallization of salts which are obtained from optically active acids and the racemic mixture, or chromatography using chiral columns. For diastereomers straight phase or reversed phase columns may be used.
The invention further includes a process for preparing a compound of the formula, the process including coupling suitably protected amino acids or amino acid analogs, followed by removing the protecting groups.
The compounds according to the general formula may be prepared in a manner conventional for such compounds. To that end, suitably Na protected (and side-chain protected if reactive side-chains are present) amino acid derivatives or peptides are activated and coupled to suitably carboxyl protected amino acid or peptide derivatives either in solution or on a solid support. Protection of the xcex1-amino functions generally takes place by urethane functions such as the acid-labile tert.-butyloxycarbonyl group (Boc), benzyloxycarbonyl (Z) group and substituted analogs or the base-labile 9-fluorenyl-methyloxycarbonyl (Fmoc). group. The Z group can also be removed by catalytic hydrogenation. Other suitable protecting groups include the Nps, Bmv, Bpoc, Aloc, MSC, etc. A good overview of amino protecting groups is given is given in The Peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, Eds., (Academic Press, New York, 1981). Protection of carboxyl groups can take place by ester formation e.g. base-labile esters like methyl or ethyl, acid labile esters like tert. butyl, or hydrogenolytically-labile esters like benzyl. Protection of side-chain functions like those of lysine can take place using the aforementioned groups. Activation of the carboxyl group of the suitably protected amino acids or peptides can take place by the azide, mixed anhydride, active ester, or carbodiimide method, especially with the addition of catalytic and racemization-suppressing compounds like 1-hydroxybenzotriazole, N-hydroxysuccinimide, 3-hydroxy4-oxo-3,4-dihydro-1,2,3,-benzotriazine, N-hydroxy-5-norbornene-2,3-dicarboximide. Also the anhydrides of phosphorus based acids can be used. See, e.g. The Peptides, Analysis, Synthesis, Biology, supra and Pure and Applied Chem. 59(3), 331-344 (1987).
It is also possible to prepare the compounds by the solid phase method of Merrifield. Different solid supports and different strategies are known see, e.g. Barany and Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, Eds., (Acad. Press, N.Y., 1980), Kneib-Cordonier and Mullen Int. J. Peptide Protein Res., 30, 705-739 (1987) and Fields and Noble Int. J. Peptide Protein Res., 35, 161-214 (1990).
Removal of the protecting groups, and, in the case of solid phase peptide synthesis, the cleavage from the solid support, can take place in different ways, depending on the nature of those protecting groups and the type of linker to the solid support. Usually deprotection takes place under acidic conditions and in the presence of scavengers. See, e.g. volumes 3, 5 and 9 of the series on The Peptides Analysis, Synthesis, Biology, supra.
Another possibility is the application of enzymes in synthesis of such compounds; for reviews see e.g. H. D. Jakubke in The Peptides, Analysis, Synthesis Biology, Vol. 9, S. Udenfriend and J. Meienhofer, Eds., (Acad. Press, N.Y., 1987).
However made, the compounds are useful for the manufacture of medicaments which have use in treating disease states involving undesired blood coagulation. In such a case the particular compound synthesized will typically be associated with a pharmaceutical carrier. Pharmaceutical carriers vary from things as relatively simple as sterilized water for injection to things as relatively complicated as microspheres and biodegradable implants.
As medicaments, the compounds are preferably administered orally, subcutaneously, topically, intranasally, intra-venously, intramuscularly or locally (e.g. via an implant). Depot administration is also possible.
The exact dose and regimen for administration of these compounds and compositions will necessarily be dependent upon the needs of the individual subject to whom the medicament is being administered, the degree of affliction or need, and of course, the judgment of the medical practitioner. In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon absorption. Illustratively however, the dosages are in the range of 0.001-100 mg per kg body weight, preferably 0.01-10 mg per kg body weight.
The medicament manufactured with the compounds may also be used as adjuvant in acute anticoagulant therapy. In such a case, the medicament is administered with other compounds useful in treating such disease states.
The compounds may also be used with implantable pharmaceutical devices such as those described in U.S. Pat. No. 4,767,628, the contents of which are incorporated by this reference. Then the device will contain sufficient amounts of compound to slowly release the compound (e.g. for more than a month).
Methods of making medicaments which can be adapted to contain the compound for enteral or parenteral administration are described in the standard reference, Gennaro et al., Remington""s Pharmaceutical Sciences, (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture), pages 1519 through 1580. Mixed with pharmaceutically suitable auxiliaries, the compounds may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection preparation, or as a spray, e.g. for use as a nasal spray.
For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.